Infants’ Folate Markers and Postnatal Growth in the First 4 Months of Life in Relation to Breastmilk and Maternal Plasma Folate

**Background:** Human milk is the sole source of folate for exclusively breastfed infants. Folate concentrations in breastmilk remain relatively constant during early lactation, while maternal folate declines. Whether breastmilk folate or maternal plasma folate are associated with infant folate status and postnatal growth is not firmly established. This study investigated these associations in 120 exclusively breastfed infants and their mothers.

**Methods:** Infants were recruited at mean age 20 (SD 3) days from a Serbian hospital and followed until 4 months. Inclusion criteria: singleton pregnancy, gestational age ≥37–≤42 weeks, birth weight 2500–4500 g, exclusive breastfeeding intended. Exclusion criteria: serious congenital diseases, >10% energy from non-breastmilk sources, maternal diabetes or vegan diet. Blood samples were collected from infants at baseline and 4 months; maternal blood and breastmilk were collected at 8 weeks postpartum. Folate markers (5-MTHF, total plasma folate, pABG, RBC-folate, tHcy) were measured using LC-MS/MS and microbiological assays. MTHFR C677T and A1298C polymorphisms were genotyped in 110 infants. Anthropometrics (weight, length, head circumference) were measured at 5 time points and converted to WHO z-scores. Statistical analyses used log10-transformed folate markers, ANCOVA with adjustment for age, weight, baseline levels, and MTHFR genotype, and GEE models for longitudinal anthropometric associations.

**Key Results:** Mean (SD) birth weight was 3446 (383) g; gestational age 39.6 (1.0) weeks. Maternal plasma 5-MTHF at 8 weeks postpartum was 20.0 (14.8) nmol/L; breastmilk 5-MTHF was 46.3 (23.3) nmol/L. Infant plasma 5-MTHF increased from 26.1 (15.5) nmol/L at baseline to 34.3 (17.6) nmol/L at 4 months. Women with breastmilk 5-MTHF >39.9 nmol/L (median) had significantly lower plasma 5-MTHF (16.6 vs. 23.3 nmol/L; p=0.015), lower total plasma folate (21.9 vs. 30.0 nmol/L; p=0.007), and lower plasma pABG (6.2 vs. 8.5 nmol/L; p=0.032) compared to women with lower milk folate. Infants of high-supplier mothers had higher plasma total folate at 4 months (adjusted p=0.049). No significant associations were found between breastmilk 5-MTHF and infant plasma tHcy (adjusted p=0.091) or RBC-folate (adjusted p=0.103). Neither breastmilk 5-MTHF nor maternal plasma folate were associated with infant weight, length, or head circumference z-scores from baseline to 4 months.

**Clinical Implications:** Higher breastmilk 5-MTHF is associated with depletion of maternal folate stores and modestly higher infant folate status, but does not appear to influence early postnatal growth. The lack of association with growth suggests adaptive mechanisms (e.g., folate-binding proteins, variable milk consumption, MTHFR polymorphisms) may buffer against low folate intake. These findings support consideration of folate supplementation during lactation, particularly in populations without folic acid fortification, to prevent maternal depletion. The study is limited by its exploratory nature, lack of maternal RBC-folate and tHcy data, and potential variability in breastmilk folate measurement.